This study identifies naturally occurring C100 lipids, whose long chain length represents a biochemical extreme that informs some general controversies related to chemotaxonomy and mycolate membrane structure. Despite genetic clustering with corynebacteria based on 16S RNA, the identification of long chain C58–C100 mycolic acids provide clear evidence for chemotaxonomic divergence of these two Segniliparus species from corynebacteria, which elaborate outer membranes with mycolates in the range of C22–36. Segniliparus mycolates are also distinguished from those in M. tuberculosis based on longer chain length and lack of oxygen-containing R groups. Thus, the chemical data presented here add to evidence that Segniliparus is unique and appropriately assigned to a new genus.
The term segnilomycolates is suggested for this class of molecules, whose distinguishing features include lack of oxygen-containing substitutions on the meromycolate chains, high degree of cis unsaturation, ultralong chain length and the large range of chain length present in a single bacterial culture. The variance in chain length of individual molecular species, which presumably form into one mycolate membrane, is unprecedently large. For example, mycolates reported here are both shorter and longer than those from Mycobacteria (C60–90) and Tsukamurella (C64–78). The observed variance is also distinctly atypical for any bacterium in the suborder Corynebacterineae. More typically, mycolyl lipids vary by 8 to 20 methylene groups, rather than 42 as seen here. Thus, intraorganismal variation in chain length raises new questions about the native conformations of mycolic acids.
The precise role for such a structural diversity of Segniliparus
α-mycolic acids is unknown, but likely influences mycolate membrane fluidity and permeability. Biochemical and structural data show that the unusual mycolates observed here form a mycolate membrane that is analogous to those found among other actinobacteria. For example, previous studies of S. rugosus
and S. rotundus
revealed brightly staining acid fast cells 
, a biochemical feature of formed mycolate membranes. Further, we provide electron micrographic evidence of an intact membrane composed of three layers of alternating electron dense and electron transparent layers. Thus, segnilomycolates likely do form a membrane and that it is not radically different in its ultrastructure compared to mycobacteria. These observations invite consideration of how the unique chemical properties of segnilomycolates might influence lipid membrane dynamics.
Detecting the long chain length and lack of oxygenated meromycolate chains in combination was initially surprising because both modifications, acting separately, would be expected to reduce membrane transition temperatures. These observations invite countervailing chemical modifications that might promote fluidity. Subsequently, we observed high degrees of cis
unsaturation, which bend meromycolate chains and have the largest effects to raise transition temperatures, which might represent a natural modification to mitigate overly tight packing 
. A simple model to accommodate longer chain length would be to produce a larger trans-membrane span. However, this simple model does not account for the C42 lipid length disparity within one organism. Further, progressively longer α’, α, and α+
mycolates are associated with one, two or three unsaturations or cyclopropyl groups, respectively (). Relatively new models of mycolate packing emphasize a possible “w” fold whereby typical mycolic acid meromycolate chains in the range of C80 use two functional groups to fold into three sections, which combined with the α-chain, appears like a “w” 
Combining these two ideas, these patterns suggest that longer chain length is associated with more highly folded molecules, rather than outstretched molecules that would span a larger transverse distance across the membrane. This mechanism might account for a homogenously thick membrane composed of mycolic acids of such divergent length. This mechanism is in agreement with prior observations of mycolic acid membrane width and density patterns, as well as simulations that predict mycolic acid folding 
. Current models predict that the α-chain and three segments of mycobacterial meromycolate chains, which are approximately C68, fold at two points of unsaturation or cyclopropanation, to create a “w”. Therefore, our data extend existing models to predict that the α+
mycolates use their α-branch, plus triply substituted, quadruply folded meromycolate chains to yield a “w +1” configuration. By this same logic, singly unsaturated α’ mycolates might represent “N” folding pattern. Future studies of membrane fluidity and in situ membrane dynamics will test these models. Last, the unusual structures seen here support further work on the role of Segniliparus
type II fatty acyl synthases and CMAS genes to better understand enzyme factors that control chain length and unsaturation